We would like to thank the entire Lazzaro lab, and especially Susan Rottschaefer, for their help in both reviewing and testing these protocols. This is a product of their cumulative expertise. Work in the Lazzaro lab is supported by grants R01 AI083932 and R01 AI064950 from the US National Institutes of Health.

1. Collect&#160;and Prepare&#160;Flies
<ol>
	<li>Rear D. melanogaster under the desired experimental conditions. Take care not to overcrowd the flies during rearing and make sure that larval densities are consistent across treatments, since environmental conditions during the larval stage can profoundly affect immune defense phenotypes during adult stage.15</li>
	<li>Collect experimental flies 0 -&#160;3 days after eclosion from the pupal case and transfer them onto fresh medium.</li>
	<li>House the collected flies at a desired temperature (temperatures between 22 &#176;C and 28 &#176;C are generally suitable) until they are aged 5 - 7 days post-eclosion. 
	NOTE: This allows sufficient time for the flies to complete metamorphosis and become mature adults, but is well before senescence begins.</li>
	<li>Sort the desired number of flies into a separate vial prior to infection, again taking care to avoid overcrowding. 
	NOTE: Only males are infected in this demonstration&#160;but is equally possible to infect females using the procedure described.</li>
</ol>
2. Culture&#160;and Prepare&#160;Bacteria
<ol>
	<li>Prepare a master plate of the chosen bacteria at least 2 days prior to infection. Streak the bacteria from a 15% glycerol stock stored indefinitely at -80 &#176;C. Store the master plate at 4 &#176;C for up to 2 weeks. Always streak the master plates directly from the frozen glycerol stock. Avoid serial passage of bacteria from plate to plate, as repeated passage in culture can cause bacteria to evolve attenuated virulence. 
	NOTE: This example makes use of Providencia rettgeri and Escherichia coli.</li>
	<li>Use the following procedure to prepare a bacterial suspension for injection.
	<ol>
		<li>Grow a 2 ml culture of bacteria by inoculating sterile medium with a single colony isolated from the master plate. Grow the bacteria to stationary phase (e.g. O/N growth at 37 &#176;C). 
		NOTE: Both P. rettgeri and E. coli grow well in Luria Broth at temperatures from 20 - 37 &#176;C with gentle shaking.</li>
		<li>Once the culture has reached stationary phase, gently pellet cells from approx. 600 &#181;l&#160;of the culture in a tabletop centrifuge (3 min at 5,000 x g), discard the supernatant, and resuspend the bacteria in approx. 1,000 &#181;l&#160;of sterile phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH = 7.4).</li>
		<li>Dilute the resuspended cells in sterile PBS to achieve an optical density (OD) appropriate for the bacterium being used, measured with a spectrophotometer as the absorbance at 600 nm. Use this suspension to infect the flies. 
		NOTE: A600 = 0.1 or 1.0 corresponds respectively to approximately 108 and 109 Providencia rettgeri or E. coli per ml. Because both live and dead bacteria contribute to optical density, it is important to harvest cultures early in stationary phase before bacterial corpses accumulate and distort the relationship between optical density and the number of viable bacteria introduced during infection.</li>
	</ol>
	</li>
</ol>
3. Infect the Flies
NOTE: As Drosophila immunity is influenced by circadian rhythm, it is important to perform infections at a similar time of day across experimental replicates.16
3.1) Using a Nanoinjector
<ol>
	<li>Prepare the glass needles for infection.
	<ol>
		<li>Prepare a glass needle by pulling a borosilicate glass capillary using a micropipette puller.</li>
		<li>Using forceps, break off the tip of the needle to create an opening of approximately 50 &#181;m diameter to allow ejection of liquid.</li>
	</ol>
	</li>
	<li>Assemble the injector.
	<ol>
		<li>Place the sealing O-ring and then the white spacer (large dimple facing outwards) onto the metal plunger.</li>
		<li>Fill the glass needle with mineral oil using a syringe with a 30 G needle.</li>
		<li>Put the filled glass needle through the collet and then place the larger O-ring around its base, about 1mm from the blunt end of the needle.</li>
		<li>Slide the needle onto the metal plunger and gently screw on the collet until secure.</li>
		<li>Eject most of the mineral oil from the injector, but make sure that there is still a small volume of oil in the needle to act as a barrier between the injector and the bacterial suspension. Make sure that there are no air bubbles within the mineral oil or bacterial suspension, or between the two liquids.</li>
		<li>Set the injector to the desired volume for injection (between 9 nl and 50 nl).</li>
	</ol>
	</li>
	<li>Generate&#160;wounding controls.
	<ol>
		<li>Fill the injector needle with sterile PBS by carefully inserting the tip of the capillary needle in a tube of the media and pressing the &#8220;fill&#8221; button on the injector. 
		NOTE: Sterile bacterial growth media can also be used a wounding control if the experiment requires injection of bacteria suspended in their growth medium. However, because bacterial growth media contains components that may stimulate the immune system or have other effects on the host, an inert carrier such as PBS is preferable.</li>
		<li>Anaesthetize the desired number of flies under a light flow of CO2.</li>
		<li>Inject the flies in the anterior abdomen on the ventrolateral surface with the sterile PBS. 
		NOTE: It is also possible to inject flies at other sites, such as the sternopleural plate of the thorax, but it is important to keep the injection site consistent within each experiment.17</li>
		<li>Place injected flies into fresh vials with new medium, laying the vials on their side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food. 
		NOTE: It is recommended to inject the PBS control flies before injecting bacteria to experimental flies so the same needle can be used for both treatments. It will not always be possible to use the same needle for an entire experiment. In that case, it may be desirable to record which needle was used with which flies and to include needle identity as an experimental factor in statistical analysis.</li>
	</ol>
	</li>
	<li>Inject&#160;the bacterial pathogen.
	<ol>
		<li>Eject the remaining sterile media from the injector and refill the same needle with the bacterial suspension.</li>
		<li>Repeat the above procedure (3.1.3), now injecting the flies with the bacterial suspension prepared in procedure 2.2.</li>
	</ol>
	</li>
</ol>
3.2) With Septic Pinprick
<ol>
	<li>Prepare the needle for pricking.
	<ol>
		<li>Melt the end of a 200 &#181;l&#160;micropipette tip and insert a 0.15 mm insect minutien pin into the molten plastic. Allow the plastic to solidify such that the pin is held in place with approximately 0.5 cm of pin extending from the plastic.</li>
	</ol>
	</li>
	<li>Generate&#160;wounding controls.
	<ol>
		<li>Anaesthetize the desired number of flies under a light flow of CO2.</li>
		<li>Prick the flies in the sternopleural plate of the thorax with the needle, avoiding the attachment sites of the wings and the legs. If necessary, gently remove flies from the minutien pin using soft forceps. 
		NOTE: It is also possible to prick the flies at other sites, such as the anterior abdomen on the ventrolateral surface, but it is important to keep the injection site consistent within each experiment.17 Pricking through the cuticle of the abdomen tends to be more difficult than pricking of the thorax and is therefore less common.</li>
		<li>Place the pricked flies into a fresh vial with new fly medium, laying the vial on its side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food.</li>
	</ol>
	</li>
	<li>Introduce&#160;the bacterial infection.
	<ol>
		<li>Anaesthetize the desired number of flies under a light flow of CO2.</li>
		<li>Prick each fly in the same location as the media controls, dipping the tip of the pin into the bacterial suspension prepared in procedure 2.2 before pricking each fly.</li>
		<li>Place the pricked flies into a fresh vial with new fly medium, laying the vial on its side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food.</li>
	</ol>
	</li>
</ol>
3.3) Assess&#160;the&#160;Infectious Dose Delivered
<ol>
	<li>Infect a set of flies as described above in either procedure 3.1 or 3.2, only instead of returning the flies to food vial to recover from anesthesia, place each fly into a microcentrifuge tube on ice.</li>
	<li>Add 250 &#181;l&#160;PBS to each tube and homogenize the flies either using a pestle or a bead beater.</li>
	<li>Plate the homogenate on an LB agar plate, either using a spiral plater or a serial dilution.
	<ol>
		<li>To plate using serial dilution, transfer each fly homogenate to the first row of a 96 well plate.</li>
		<li>Fill each well of the remaining rows with 90 &#181;l&#160;of PBS.</li>
		<li>Using a multichannel pipette, take 10 &#181;l&#160;from the first row containing fly homogenate and dispense into the second row.</li>
		<li>Pipette up and down at least 10 times to thoroughly mix, and then take 10 &#181;l&#160;and transfer to the third row. Repeat this procedure using the remaining rows.</li>
		<li>Starting from the bottom row (most dilute bacteria suspension), use the multichannel pipette to take 10 &#181;l&#160;from each well and deposit on an LB plate, taking care that the samples are dispensed as discrete spots that do not touch each other. Repeat until all wells have been sampled from each row, dispensing them in descending order of dilution on the LB plate.&#160;</li>
		<li>Leave the plate at RT&#160;until the spots have completely soaked into the LB plate. 
		NOTE: Drying the LB plate for a few days at RT prior to use is recommended to ensure that the liquid is readily absorbed, minimizing the chances of accidental contact between sample spots.</li>
	</ol>
	</li>
	<li>Incubate the plates O/N, taking care not to overgrow the plates so colonies remain small and discrete. 
	NOTE: Depending on the bacterial growth medium and incubation temperature used, colonies derived from the fly&#8217;s endogenous gut microbiota may eventually appear on the plate. However, most bacteria used for pathogenic infection grow much faster than the gut microbiota on LB agar at 37 &#176;C.</li>
	<li>Remove plates from the incubator when the experimental bacteria have&#160;grown visible colonies (typically 8 - 12 hr), but before Drosophila gut microbiota colonies appear (approximately 36 hr).&#160; For slow growing experimental bacteria, use a selective antibiotic in the LB agar to remove any colonies from the gut microbiota.</li>
	<li>Count the number of colonies for each homogenate.
	<ol>
		<li>For spiral plates, count the colonies that grow using an automated colony counter that can estimate the bacterial load per ml&#160;of homogenate based on the number and position of the colonies on the plate. 
		NOTE: Spiral counts are most accurate when the bacterial concentration is in the range of 5 x&#160;102 to 5 x 104 bacteria per ml.</li>
		<li>For spot plates, manually count the colonies for each fly from whichever dilution contains 30 - 300 colonies and calculate the number of bacteria per ml&#160;of original homogenate.</li>
	</ol>
	</li>
</ol>
4. Characterize Survivorship of Infection
<ol>
	<li>Assay&#160;mortality post-injection.
	<ol>
		<li>Place infected flies and wounded controls in fresh vials and maintain in an incubator at a desired temperature (25 &#176;C with a 12:12 light:dark cycle is recommended). Do not put more than 15 flies into a single vial&#160;as it becomes difficult to count more than 15 living flies.</li>
		<li>Count the number of living and dead flies each day, or more frequently if appropriate.</li>
		<li>In order to maintain food quality, flip the flies to new food on a regular schedule.
		<ol>
			<li>If the vial contains only males, transfer them to fresh vials every three days. If the vial contains females, transfer to fresh vials every two days because larval progeny will liquefy the food, increasing the risk that adult flies may get stuck and resulting in overestimated rates of mortality due to infection.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Statistically analyze&#160;the data.
	<ol>
		<li>Plot survival as a Kaplan-Meier curve, which indicates the probability that an individual will survive to a measured time-point post injection.18 For survival curves that do not cross, perform pair-wise comparisons using a Log-Rank Test (also called Mantel Cox Test) or perform more complex analyses using a Cox Proportional Hazards Model, which can incorporate several factors and their interactions. For survival curves that cross, perform a stratified Cox analysis.17</li>
	</ol>
	</li>
</ol>
5. Assay Bacterial Load Post-infection
<ol>
	<li>Measure&#160;bacterial load.
	<ol>
		<li>At a prescribed time post-injection, repeat the procedure used to assess infectious dose to determine bacterial load over the course of the infection (see protocol 3.3). 
		NOTE: If using the spiral plater, dilute homogenates so that the bacterial suspension falls within a range of 1,000 - 100,000 bacterial per ml.</li>
	</ol>
	</li>
	<li>Analyze&#160;the data.
	<ol>
		<li>If bacterial load is non-normal, either transform the data to better approximate a normal distribution or analyze the data using non-parametric statistical analysis (e.g. Mann-Whitney U test). Use a Box-Cox analysis to find the optimal transformation; natural log or base -10 log transformations are often effective.19</li>
		<li>If the data are sufficiently normally distributed and there are only two treatments being contrasted, perform a t-test to determine whether the two treatments result in different mean bacterial loads. If there are multiple experimental variables or other potentially predictive factors, use Analysis of Variance (ANOVA) to determine which factors are significant predictors of bacterial load.19</li>
	</ol>
	</li>
</ol>
6. Assay&#160;Transcriptional Activation of Immune System Genes
<ol>
	<li>To coarsely visualize immune activation after infection, infect transgenic flies that express green fluorescent protein (GFP) under the control of a promoter from an antimicrobial peptide gene as previously described.18</li>
	<li>View the infected flies under a fluorescence-capable microscope; GFP induction should become visible in the fat body within the first 6 - 10 hr after infection.</li>
	<li>For more precise quantification of immune activation, use quantitative PCR on cDNA template (qRT-PCR) to measure the abundance of antimicrobial peptide gene transcripts. 
	NOTE: The expression of immune genes can be measured at any time after (or before) infection. Generally speaking, induction of immune gene expression begins approximately 4 hr after injection and increases over the next 24 hr.
	<ol>
		<li>Anesthetize flies using CO2.</li>
		<li>Place 15 flies into a microfuge tube for each RNA extraction. 
		NOTE: It is recommended to collect at least three replicate samples for each treatment condition.</li>
		<li>Extract RNA from the flies and generate first-strand cDNA using any of a number of standard procedures or commercial kits. Store RNA at -80 &#176;C. Store cDNA at -20 &#176;C for periods of a few weeks&#160;and at -80 &#176;C for long term storage. Prior to RNA extraction, store flies at -80 &#176;C.</li>
		<li>Perform quantitative PCR (qPCR) on target genes of interest using the cDNA as a template. Refer to Table 1 for primer sequences to amplify the antibacterial peptide genes Diptericin A, Drosomycin, Defensin, Attacin A, and Metchnikowin as well as the housekeeping control gene rp49 (also known as rpL32). 
		NOTE: The genes encoding the antimicrobial peptides Diptericin A and Drosomycin provide very good readouts of D. melanogaster immune activity induced mainly through the Imd and Toll pathways, respectively. In order to control for sample-to-sample variation in RNA yield and the efficiency of reverse transcription, standardize the recorded expression of target genes against a reference housekeeping gene whose expression is not expected to change with infection such as rp49 (also known as rpL32).</li>
		<li>Analyze the data.
		<ol>
			<li>For rough approximation of expression differences, calculate the ratio of the starting quantity (SQ) value obtained from the test gene (e.g., Diptericin A) relative to the SQ value obtained from the reference gene (e.g., rp49) for each sample (calculated as SQ-DptA/SQ-rp49). Scale these ratios to a baseline control treatment&#160;such as an uninfected handling control. Arbitrarily set the control expression level equal to 1.0 and visualize experimental treatments as relative fold changes. Estimate the SQ value for each gene against a standard curve generated from a dilution series of known-quantity cDNA. 
			NOTE: Statistical analysis of ratios can be complicated, so this approach is better for quick approximation than for rigorous analysis. If PCR efficiency is low, design new primers to improve PCR kinetics if possible. For qPCR reactions with near-perfect efficiency (a doubling of PCR product every cycle), the threshold cycle (CT) can be substituted for the SQ value.</li>
			<li>For simple experiments with optimally efficient amplification, data may be analyzed using the &#916;&#916;CT method.19 For each sample, subtract the CT value of the reference gene (e.g., rp49) from the CT of the test gene (e.g., Diptericin A) to yield a &#916;CT. Then subtract the &#916;CT of the baseline control from the &#916;CT for each experimental sample to yield a &#916;&#916;CT value for each sample; this &#916;&#916;CT is indicative of relative differences in gene expression levels between treatments, where 2(-&#916;&#916;CT) approximates the fold change in expression. 
			NOTE: This analysis can badly misestimate fold-change in expression of the target gene if the PCR of either the test gene or the reference gene has low efficiency.</li>
			<li>For the most rigorous analysis, conduct analysis of variance (ANOVA) using the estimated expression of the test gene (e.g.&#160;Diptericin A) as the response variable and estimated expression of the control gene (e.g.&#160;rp49) and other experimental factors as explanatory variables. 
			NOTE: This approach is relatively robust to inefficiency in PCR amplification and allows analysis of more complicated experimental designs.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biometry. , (1995).</li><li>Frydman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+bacterial+infection+in+Drosophila.">Wolbachia bacterial infection in Drosophila.</a> Journal of visualized experiments JoVE. (2), 158 (2007).</li><li>Kuo, T. -. H., Handa, A., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+the+immune+response+and+sleep+in+Drosophila.">Quantitative measurement of the immune response and sleep in Drosophila.</a> Journal of visualized experiments JoVE. (70), e4355 (2012).</li><li>Vodovar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+host+defense+after+oral+infection+by+an+entomopathogenic+Pseudomonas+species.">Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (32), 11414-11419 (2005).</li><li>Nehme, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+bacterial+intestinal+infections+in+Drosophila+melanogaster.">A model of bacterial intestinal infections in Drosophila melanogaster.</a> PLoS pathogens. 3 (11), e173 (2007).</li><li>Jaenike, J., Polak, M., Fiskin, A., Helou, M., Minhas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecific+transmission+of+endosymbiotic+Spiroplasma+by+mites.">Interspecific transmission of endosymbiotic Spiroplasma by mites.</a> Biology. 3 (1), 23-25 (2007).</li><li>Kanbar, G., Engels, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+and+bacterial+infection+of+wounds+in+honey+bee+(+Apis+mellifera)+pupae+punctured+by+Varroa+mites.">Ultrastructure and bacterial infection of wounds in honey bee ( Apis mellifera) pupae punctured by Varroa mites.</a> Parasitology research. 90 (5), 349-354 (2003).</li></ol>

None of the authors have competing interests or conflicting interests.

The procedure described here yields rigorous and high quality infection of Drosophila melanogaster. The illustrated examples primarily focused on infection with Providencia rettgeri and E. coli, but the protocol is highly adaptable and can be applied to infections with diverse bacteria over a range of host rearing and maintenance conditions.
The details of an optimal experimental approach will depend on the bacterium used for infection, the genotype of the host, and the overall experimental conditions. It is strongly recommended to pilot test any new experimental conditions before initiating more ambitious projects. A good starting point is to test three infection doses over a 100-fold range. Highly virulent pathogens are often best introduced at very low infectious doses, on the order of 10 - 100 bacterial cells per fly. More moderate pathogens can be injected at higher doses of around 1,000 bacteria per fly, and non-pathogens may be injected at doses as high as 10,000 bacteria per fly. It is often instructive to define the kinetics of novel infections by tracking pathogen load, host mortality, and immune system activity over a longitudinal time series. Because measurement of pathogen load and host gene expression are destructive assays, it is necessary to infect distinct flies at the outset of the experiment for every time point that is to be measured.
When deciding whether to use pinprick or microcapillary-based injection, it is important to note there are advantages and limitations to each approach. Capillary injection introduces a volume of liquid into the fly, which both modestly increases turgor pressure and introduces salts or other molecules that are suspended or dissolved in the carrier. Capillary injection also requires access to an injection facility or purchase of the required equipment. Septic pinprick requires no special equipment and introduces negligible media into the fly, and is typically more efficient for infecting large numbers of flies. However, pinprick infections do not allow the precise control over infection dose that can be achieved with capillary injection. The present protocol focused on an injection apparatus that mechanically regulates injection volume, but there are also injection systems based on discrete pulses of pressurized air.20,21 These are typically more expensive than the apparatus featured here and require calibration of the air pulse to each needle in order to ensure consistent injection volumes.
There is considerable debate but very little data on how flies become systemically infected with bacteria in the wild. Some investigators posit that the majority of natural infections occur when Drosophila ingest pathogenic bacteria&#160;and the bacteria are subsequently able to escape the gut to&#160;establish a systemic infection. However, there are very few bacteria known to be able to cross the gut of D. melanogaster, and those that do have this ability are highly lethal to flies22,23. An alternative theory is that flies regularly sustain cuticular injuries through escape from failed predation attempts or attack by ectoparasitic mites. This hypothesis is supported by the frequent collection of wild D. melanogaster bearing melanization spots that are indicative of healed wounds (unpublished observations). Mites have been shown to transmit bacterial infections in Drosophila24 and wounds left by mites can be secondarily infected by bacteria in honey bees25. However, the frequency in nature of mite-driven or otherwise opportunistic infection of D. melanogaster through cuticle breaches is not known. The protocol described here allows introduction of bacteria directly into the hemolymph through quantitative injection which bypasses any epithelial barriers or behavioral immunity. Methods for feeding pathogenic bacteria to D. melanogaster have been described in Vodovar et al.22 and Nehme et al.23.
Many entomopathogenic bacteria pose little or no human health risk, allowing researchers to work with them comfortably. Furthermore, very few bacteria have the capacity to infect Drosophila on contact without experimental intervention, so the risk of &#8220;epidemic&#8221; spread of bacterial infection through a laboratory via contaminated surfaces or escaped flies is generally very low. Nonetheless, it is advisable to ensure that adequate containment measures are in place to prevent infected flies from escaping and for recapturing any escaped flies. The laboratory should be outfitted at a biological safety level commensurate with that of the pathogens being used, and standard best practices in microbiology should be employed.
The experimental infection method described here allows infections of Drosophila melanogaster with any dose of any arbitrary bacterium. Once infection has been established, it is straightforward to measure the kinetics of bacterial proliferation or clearance, to track host mortality, and to assay induction of the host immune system. Infected flies can easily be subjected to other phenotypic assays, including tests of physiological functions that may shape or be shaped by the infection. The procedures described are inexpensive, require relatively little specialized equipment, and are easily learned, making them amenable for use in diverse projects across a breadth of research and teaching labs.

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Drosophila are inexpensive and easy to rear, are highly amenable to experimental manipulation, and are backed by an extensive scientific community that has developed a broad array of research tools. Many aspects of innate immunity are conserved between insects and mammals, including signal transduction mediated by Toll-like receptors and NF-kB family transcription factors, JAK/STAT signaling, and JNK pathway responses.1,2 The function of these genes and pathways can be queried in D. melanogaster using mutations or RNAi knockdowns that increase or decrease pathway activities.3&#8211;6 Additionally, Drosophila can be used to study the physiological consequences of infection and disease, including in the context of evolutionary life history theory.7&#8211;9 All such studies, however, depend on the ability to reliably infect experimental flies under defined treatment conditions. The procedure described here presents a methodological framework for delivering robust and repeatable bacterial infections to Drosophila melanogaster and subsequently measuring infection severity and quantifying the host immune response.
Drosophila can be naturally and experimentally infected by a wide variety of parasites and pathogens, including bacteria, fungi, viruses, nematodes and parasitoid wasps. The current protocol is focused on delivering systemic bacterial infection. Many different bacteria can be used to infect flies, and the experimenter&#8217;s choice should be based on the precise scientific questions being asked. For example, human clinical isolates may be employed to study bacterial virulence mechanisms10, or ecologically relevant isolates may be preferred for evolutionary study.11 Some bacteria are competent pathogens of D. melanogaster, proliferating upon infection and causing host sickness or death. Other bacteria are effectively managed by the host immune system and cleared within a few days. In this demonstration, Providencia rettgeri will be used as a proliferative pathogen that can cause host mortality and persists in surviving hosts. Escherichia coli will be used as a non-pathogen that is cleared by the host immune system.
Infection will be established by introduction of bacteria directly into the body cavity of the fly. This approach bypasses epithelial barriers and protective behaviors, allowing investigation of systemic infection irrespective of the natural mode of transmission. There are two primary methods for experimentally establishing systemic infection. In the first, a nanoinjector and pulled glass capillary needles are used to inject a precise number of bacteria into the fly. This method has the advantages of allowing a large dynamic range of infection doses and of being quantitatively highly repeatable. The second approach is to deliver infection with a septic pinprick. This approach has the advantages of being rapid and requiring no special equipment. Once the infections are established, it becomes possible to measure systemic pathogen load, host mortality, and inducible immune system activity. Of course, any number of additional phenotypes could conceivably be measured in infected D. melanogaster, including post-infection fecundity12, learning ability13, metabolic status14, or virtually any other trait that can be imagined.

<table border="0" cellpadding="0" cellspacing="0" width="815">
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			<td height="21" style="height:21px;width:399px;">Reagent</td>
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</table>

systemic bacterial infection and immune defense phenotypes in drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

This section illustrates results that can be obtained after bacterial infection of Drosophila melanogaster. Figure 1 shows that infection dose varies with optical density of the bacterial suspension used for injection, and that the dose delivered can be reliably estimated by homogenizing and plating flies immediately after injection. As illustrated in Figure 2, different pathogens can cause different levels of host mortality (Figure 2A) and host mortality can be dose-dependent (Figure 2B). Importantly, this protocol allows for different types of infections to be achieved: Providencia rettgeri can cause a chronic, sub-lethal infection that persists for 20 days or longer (Figure 3A). However, other bacteria like Escerichia coli will mostly be cleared by the host fly within six hours post-infection (Figure 3B). Induction of the immune system can be estimated through RNA isolation and subsequent qRT-PCR of antibacterial peptide transcripts (Figure 4A and B). Analogously but less quantitatively, flies expressing GFP under the control of antimicrobial peptide gene promoters can be used to visualize induction of the immune system (Figure 4C).
<img alt="Figure 1" src="/files/ftp_upload/52613/52613fig1.jpg" /> 
Figure 1: Determining Infectious Dose. Flies were injected with 50 nl&#160;of bacterial suspensions covering a range of optical densities (0.0001 - 0.05). The flies were immediately homogenized and plated to determine the number of bacterial introduced by the injection. Initial bacterial load strongly correlates with initial OD injected (r2 = 0.96)
<img alt="Figure 2" src="/files/ftp_upload/52613/52613fig2.jpg" /> 
Figure 2: Survival after Injection with Bacterial Pathogens. Five to seven day old males were injected with 50 nl&#160;of either bacterial suspension or sterile media and monitored for survival. (A) Wild type flies were injected with approximately 5,000 bacteria from of one of three different species. The rate of host mortality depends heavily on the bacterial species used for infection. (B) Wild type flies were injected with three different doses of Enterococcous faecalis - 50, 500, and 5,000 bacteria per fly. Flies die more quickly when infected with higher infectious doses (C) An immune system mutant and its wild-type isogenic control line were injected with approximately 500 Burkholderia cepacia. A pairwise Log-Rank comparison shows that the wild-type fly survives the infection significantly better than the mutant (&#967;2&#160;= 59.02, df = 1, p &#60;0.0001).
<img alt="Figure 3" src="/files/ftp_upload/52613/52613fig3.jpg" /> 
Figure 3: Bacterial Load after Injection. Flies were injected with approximately (A) 5,000 P. rettgeri (B) 3,400 E. coli. Bacterial load was determined immediately after injection and at various subsequent time points. Each data point represents the bacterial load of a single fly. E. coli is quickly cleared from challenged hosts while P. rettgeri persists for the remainder of the host&#8217;s life,.
<img alt="Figure 4" src="/files/ftp_upload/52613/52613fig4.jpg" /> 
Figure 4: Induction of Immune Gene Expression after Injection. (A) Flies were injected with 50 nl&#160;of P. rettgeri or sterile PBS in either the abdomen or thorax, or were left unmanipulated aside from CO2 anesthesia. Six hours after infection, flies were collected for RNA isolation and expression of the Diptericin A gene was determined using qRT-PCR. (A) Expression levels are graphed as the ratio of Diptericin A transcript to rp49 transcript and scaled such that the CO2 control is defined as having an expression level of 1. The bars represent the mean and standard error of ratios from each condition (n = 4). (B) Expression levels are graphed as a 2&#916;&#916;CT with the average CT value from CO2 aesthesia control flies used as the standard uninduced condition. The bars represent the mean and standard error of &#916;&#916;CT from each condition (n = 4). While there is no difference in transcript induction due to site of injection, comparison of panels A and B shows how the &#916;&#916;CT method can potentially overestimate induction levels. (C) Flies expressing GFP under control of the Diptericin A promoter were injected with 50 nl&#160;of P. rettgeri (OD = 0.1) and then imaged 7 days later. The GFP panel shows expression of GFP in the abdominal fat body of infected flies, indicating activation of the Diptericin A promoter in bacterially-infected flies but not media-injected controls.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gene</td>
			<td>Forward</td>
			<td>Reverse</td>
		</tr>
		<tr>
			<td>rp49 (also called rpL32)</td>
			<td>5&#8217; AGGCCCAAGATCGTGAAGAA 3&#8217;</td>
			<td>5&#8217; GACGCACTCTGTTGTCGATACC 3&#8217;</td>
		</tr>
		<tr>
			<td>Diptericin A</td>
			<td>5&#8217; GCGGCGATGGTTTTGG 3&#8217;</td>
			<td>5&#8217; CGCTGGTCCACACCTTCTG 3&#8217;</td>
		</tr>
		<tr>
			<td>Drosomycin</td>
			<td>5&#8217; CTGCCTGTCCGGAAGATACAA 3&#8217;</td>
			<td>5&#8217; TCCCTCCTCCTTGCACACA 3&#8217;</td>
		</tr>
		<tr>
			<td>Defensin </td>
			<td>5&#8217; GAGGATCATGTCCTGGTGCAT 3&#8217;</td>
			<td>5&#8217; TCGCTTCTGGCGGCTATG 3&#8217;</td>
		</tr>
		<tr>
			<td>Attacin A</td>
			<td>5&#8217; CGTTTGGATCTGACCAAGG 3&#8217;</td>
			<td>5&#8217; AAAGTTCCGCCAGGTGTGAC 3&#8217;</td>
		</tr>
		<tr>
			<td>Metchnikowan</td>
			<td>5&#8217; AACTTAATCTTGGAGCGATTTTTCTG 3&#8217;</td>
			<td>5&#8217; ACGGCCTCGTATCGAAAATG 3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 1: Primers for qRT-PCR.

Watch this Scientific Journal Video about Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster at JoVE.com

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

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Immunology and Infection

24.5K Views.  Cornell University. Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Video: Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...